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Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells.

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.

4-Butyrolactone↗

A new alpha-methylene gamma-lactone sesquiterpene from Hedyosmum arborescens.

A new sesquiterpene lactone, 7alpha,10alpha-epoxy-1alpha(H),5alpha(H)-guaia-3,11(13)-dien-8alpha,12-olide, was isolated from the leaves of Hedyosmum arborescens. The structure of this unusual 7,10-epoxy-guaianolide was determined by spectroscopic methods, particularly one- and two-dimensional 1H and 13C NMR.

Magnetic Resonance Spectroscopy↗

Effect of temperature and glucose concentration on the N-butanoyl-L-homoserine lactone production by Aeromonas hydrophila.

N-butanoyl-L-homoserine lactone (C4-HSL) production by Aeromonas hydrophila 519 has been established. C4-HSL production at 22 and 30 degrees C was found after 20-24 h of incubation corresponding to a population density of ca. 10(8)-10(9) cfu/ml, respectively. Reduced C4-HSL production was noted after 72 h of incubation at 12 degrees C when the culture reached ca. 10(9) cfu/ml. No C4-HSL production was detected at 37 degrees C, although a dense population was obtained. In LB broth supplemented with 0.1% and 0.5% glucose, C4-HSL production was noted whereas with 1% glucose no C4-HSL was detected although a high colony count was obtained. In the latter culture residual levels of glucose (0.65%) were found after 43 h whereas in the 0.1% and 0.5% supplemented LB, glucose was quickly consumed which may have stimulated C4-HSL production. In conclusion, the present study shows an effect of environmental conditions (temperature, glucose concentration) on the C4-HSL production and warrants further investigation to elucidate the effect of external conditions on production of AHL signal molecules to reveal the relevance of quorum sensing in, e.g. foods.

4-Butyrolactone↗

Influence of food system conditions on N-acyl-L-homoserine lactones production by Aeromonas spp.

Eleven of 13 Aeromonas strains were shown to produce AHLs. Results of TLC showed that N-butanoyl-L-homoserine lactone (C4-HSL) was the main AHL produced in LB medium at 30 degrees C. The influence of different carbon sources, temperature, pH values and salt concentrations on AHL production was determined in eight A. hydrophila and one A. caviae strain. Additionally a quantitative study of C4-HSL production by A. hydrophila strain 519 under different conditions was performed. Positive results were found in the AHL induction assay for some Aeromonas strains in cultures in LB agar incubated at 12 degrees C after 72-96 h. The induction of the sensor strains by Aeromonas spp. occurred in LB medium supplemented with all carbon sources in a concentration of 0.5%. The production of C4-HSL by A. hydrophila 519 was found until 3.5% (w/v) of NaCl. For pHs close to the neutrality the C4-HSL production by A. hydrophila was evident after 24-48 h of incubation. A. hydrophila 519 produced C4-HSL under anaerobic conditions. Also, the AHL production by Aeromonas strains was studied in simulate agar of shrimp, fish and some vegetables. The production of AHLs was evident by almost all the test strains in shrimp simulated agar. In fish agar only for one of three fish species tested, positive results were found. Induction assay in vegetables simulated agar showed principally negative results, probably because of the presence of inhibitory compounds in these vegetables.

4-Butyrolactone↗

N-acylhomoserine lactone-dependent cell-to-cell communication and social behavior in the genus Serratia.

Members of the genus Serratia are increasingly responsible for nosocomial infections, the treatment of which may be complicated by the appearance of multi-antibiotic-resistant strains. Some but not all Serratia strains and species produce N-acylhomoserine lactones (AHLs), and possess luxR and luxI homologous genes. Phylogenetic comparisons have provided evidence for the lateral transfer of these quorum-sensing systems, and in at least one strain of S. marcescens, transfer via a complex transposon has been experimentally demonstrated. AHL-dependent quorum sensing in Serratia controls population surface migration, biofilm development, the biosynthesis of a carbapenem antibiotic and production of the red pigment, prodigiosin. Serratia also possesses LuxS and produces autoinducer-2 (AI-2) which appears to function as a second quorum-sensing system controlling many of the same phenotypes as the LuxR/AHL systems.

4-Butyrolactone↗

The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway.

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.

4-Butyrolactone↗

N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit.

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.

4-Butyrolactone↗

A simple, rapid, sensitive method detecting homoserine lactone (HSL)-related compounds in microbial extracts.

A simple, rapid, sensitive microtiter plate method detecting N-acyl homoserine lactone (HSL)-related compounds was established using an Agrobacterium tumefaciens strain harboring a traG::lacZ/traR reporter gene responsive to HSLs. This strain did not produce its own HSL, but the traG::lacZ reporter gene was induced only when its transcription activator TraR detected a cognate exogenous HSL. Therefore, the assay was expected to be highly specific for HSL-related compounds. Induction of the reporter gene, leading to production of beta-galactosidase enzyme, was measured by using two different beta-galactosidase substrates, X-gal and Galacton-Star, for colorimetric and chemiluminometric detection, respectively. The screen was validated in both the 96-well and 384-well plate formats, and extracts derived from 696 different microbial isolates, mostly unidentified actinomycetes isolated from diverse locations, were tested. Crude extracts of 81 (11.64%) cultures tested positive for HSL-related compounds, and an additional 34 (4.8%) crude extracts showed a moderate to weak signal for HSLs. Data from the fractionated samples, however, suggested a much higher prevalence of HSL signals in these extracts. Of 144 crude extracts fractionated into 10 individual samples at a 10x concentration, 72 (50%) cultures tested positive for HSLs. Six cultures were active only in the crude extract, 18 were active both in crude and one or more of their fractions, and an additional 48 were active in just one or more of their fractions. This finding may be the first to suggest such a high prevalence of HSL-signals found in nature, and a large number of actinomycetes in our collection appeared to produce HSL-related compounds.

4-Butyrolactone↗

Direct quantification of N-(3-oxo-hexanoyl)-L-homoserine lactone in culture supernatant using a whole-cell bioreporter.

The autoinducer N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) plays a significant role in the quorum-sensing system of the marine bacterium Vibrio fischeri. Upon forming a transcriptional activation complex with LuxR, 3-oxo-C6-HSL induces transcription of the luxICDABEG operon, leading to the increased production of both the 3-oxo-C6-HSL synthase (LuxI) and the bioluminescent proteins. In order to quantitatively analyze this regulatory mechanism, a novel approach was developed to measure 3-oxo-C6-HSL concentrations in V. fischeri cell culture supernatant. A bioluminescent strain of Escherichia coli that responds to 3-oxo-C6-HSL was used as a bioreporter. Although a linear response of the bioreporter to exogenously added synthetic 3-oxo-C6-HSL was found over several orders of magnitude, we show that bioreporter performance was dramatically impacted by variations in the supernatants using samples from a V. fischeri LuxI- strain. However, when maintained in the same supernatant background, the normalized peak bioluminescence maintained a linear response to 3-oxo-C6-HSL concentrations. Therefore, a standard additions technique was developed in which a known concentration of 3-oxo-C6-HSL was added to supernatant samples from wild-type V. fischeri cultures, and the incremental increase of the normalized peak bioluminescence relative to the untreated sample was determined. The concentration of 3-oxo-C6-HSL in the supernatant of the unknown sample was then quantified from the slope of the response between the normalized bioluminescent peaks with and without the addition of 3-oxo-C6-HSL. Advantages of this method are that it is rapid, does not require concentration or extraction, uses a small sample volume (ca. 2 ml), and accounts for effects caused by the composition of the supernatant. Furthermore, the findings can be broadly applicable to other bioreporter systems involving variable background conditions.

4-Butyrolactone↗

Correlation between production of acyl homoserine lactones and proteases in an Aeromonas hydrophila aroA live vaccine.

Aeromonas hydrophila is a pathogen that causes disease in a wide range of homeothermic and poikilothermic hosts due to its multifactorial virulence. We have previously described the characterisation and use of an auxotrophic aroA mutant of the A. hydrophila AG2 strain as a live attenuated vaccine against A. hydrophila infections in rainbow trout (Oncorhynchus mykiss). In this study we report the expression of extracellular proteolytic activities and of quorum-sensing molecules by this mutant grown under different culture conditions, and in vaccine inocula. The aroA strain expresses extracellular proteases efficiently during in vitro growth and this ability is retained in vaccine inocula that were prepared by washing the bacterial cultures and resuspending the cells in phosphate-buffered saline. Since proteases are considered to be major bacterial antigens, the expression of these enzymes in the live attenuated vaccine may contribute to the superior protection afforded by these kind of vaccines. On the other hand, the production of serine- and metalloprotease activities in A. hydrophila has been described as controlled in a cell density-dependent fashion, through a mechanism known as quorum sensing. A microtiter method was developed that allowed correlation of the production of quorum-sensing molecules and of proteases produced by the aroA strain during in vitro growth and in the vaccine inocula. The production of both products was related to the type of culture medium and conditions used to grow the aroA mutant, whereas there was no correlation between the concentration of acyl homoserine lactones and protease production.

4-Butyrolactone↗

The safety-net story about macrocyclic lactone heartworm preventives: a review, an update, and recommendations.

A number of safe, effective, and convenient heartworm preventatives are currently available for virtually all canine and feline pets. Yet, a 2001 survey of over 18,000 veterinary clinics in the United States identified more than 240,000 dogs and 3000 cats infected with Dirofilaria immitis. This high level of owner compliance failure is alarming. Prolonged administration of some of the macrocyclic lactone (ML) preventatives kills young larvae, older larvae, "immatures," young adults, and/or old adults. Efficacy of 95% or more requires dosing for 9-30 months, with older worms being more difficult to kill. Of the various MLs, ivermectin (IVM) has the most potent safety-net and adulticidal activity, milbemycin oxime has the least, and selamectin and moxidectin injectable lie somewhere in between. The unique effects of IVM are related to the age of the heartworms at initiation of treatment. The earlier treatment is started, the more stunted and smaller the worms and the shorter their survival time. Conversely, the later treatment is started, the longer the worms live, and the more likely the dog will be antigen- and microfilariae-positive. Drug effects do not appear to be enhanced by increasing the dosage or administering at shorter intervals, and it appears that continuous monthly treatment is needed to produce the full effects of the drug. The American Heartworm Society (AHS) recognizes the safety-net (or reach-back effect) and adulticidal properties of some MLs, particularly IVM. The AHS 2003 (American Heartworm Society, 2004. 2003 Updated guidelines for the diagnosis, prevention, and management of heartworm (Dirofilaria immitis) infection in dogs. In: McCall, et al., (Eds.), Proceedings of the Symposium Session on Recent Advances in Heartworm Disease, The 19th International Conference of the World Association for the Advancement of Veterinary Parasitology, New Orleans, LA, 10-14 August, 2003. Vet. Parasitol. 125, 105-130) canine guidelines state that it is beneficial to administer prophylactic doses of IVM before treatment with melarsomine. Results of laboratory studies suggest that less active dogs are at low risk of severe thromboembolism and death. However, heartworm-positive working dogs might be more at risk. Worsened radiographic and echocardiographic images in a client-owned dog given IVM monthly for 2 years with greatly restricted exercise suggests that such treatment of dogs with clinical, radiographic, and/or echocardiographic evidence of heartworm disease as well as for asymptomatic working dogs is contraindicated. Furthermore, until further data are available, such treatment of even the less active asymptomatic dog should be administered only with much caution and with examination by a veterinarian at least once every 4-6 months. IVM clearly provides potent "safety-net" activity against older larvae, immatures, and young adults in cases of owner compliance failure, even when the owner and veterinarian are not aware that the animal is infected, and offers much promise as a unique "soft-kill" treatment for young, and possibly older adult heartworms, with reduced risks.

Animals↗

Suppression by a sesquiterpene lactone from Carpesium divaricatum of inducible nitric oxide synthase by inhibiting nuclear factor-kappaB activation.

Excessive nitric oxide (NO) produced by inducible NO synthase (iNOS) acts as a causative regulator in various inflammatory disease states. Carpesium divaricatum has been used in Korean traditional herbal medicine for its antipyretic, analgesic, vermifugic, and anti-inflammatory properties. We investigated the molecular mechanism for the suppression of lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production in RAW 264.7 macrophages by the sesquiterpene lactone 2beta,5-epoxy-5,10-dihydroxy-6alpha-angeloyloxy-9beta-isobutyloxy-germacran-8alpha,12-olide (C-1), which has been identified recently as a new compound from C. divaricatum. C-1 decreased NO production in LPS/IFN-gamma-stimulated RAW 264.7 cells in a concentration-dependent manner, with an IC50 of approximately 2.16 microM; however, it had no direct effect on the iNOS activity of fully LPS/IFN-gamma-stimulated RAW 264.7 cells. Furthermore, treatment with C-1 led to a decrease in iNOS protein and mRNA. These effects appear to be due to inhibition of nuclear factor-kappaB (NF-kappaB) activation through a mechanism involving stabilization of the NF-kappaB/inhibitor of the kappaB (I-kappaB) complex, since inhibition of NF-kappaB DNA binding activity by C-1 was accompanied by a parallel reduction of nuclear translocation of subunit p65 of NF-kappaB and I-kappaBalpha degradation. Taken together, the results suggest that the ability of C-1 to inhibit iNOS gene expression may be responsible, in part, for its anti-inflammatory effects.

Animals↗

Effect of sesquiterpene lactones on the expression of the activation marker CD69 and of IL-2 in T-lymphocytes in whole blood.

We used flow cytometry to investigate the inhibitory effect of sesquiterpene lactones (SLs) on T-cell activation measured by the expression of its early marker CD69, and on interleukin (IL)-2, a mediator of activation, in whole blood. SLs are biologically active compounds found especially in plants from the Asteraceae family. Overnight treatment of blood with these substances led to the inhibition of CD69 and IL-2 expression. Interestingly, bifunctional SLs showed a weaker activity than monofunctional substances, which is in contradiction with the data obtained so far, using other biological test systems. Additionally, SLs did not completely inhibit CD69 or IL-2 expression. We also determined their toxicity and observed only a low effect. Up to now, studies on cytotoxicity have only been performed using cultured cell lines. From these results it may be supposed that these natural compounds preferentially show toxic effects towards transformed cell lines. Altogether, the results demonstrated that SLs effectively inhibit the activation of the T-lymphocyte response in whole blood and proved the utility of a whole blood system in studying their biological effects.

Antigens, CD↗

Rapid access to uronic acid-based mimetics of Kdn2en from D-glucurono-6,3-lactone.

A concise route to novel mimetics of Kdn2en, based on delta4-uronic acids, from D-glucurono-6,3-lactone is presented. Uronic acid-based mimetics in which an aliphatic ether (O-glycoside), a thioether (S-glycoside), or acetamide takes the place of the natural C-6 glycerol sidechain of the sialic acid were synthesized from the key intermediate, methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate bromide.

Glucuronates↗

Inhibition of Ca2+-independent phospholipase A2 by bromoenol lactone attenuates prostaglandin generation induced by interleukin-1 beta and dibutyryl cAMP in rat mesangial cells.

Cytokine-induced prostaglandin generation in rat mesangial cells has been suggested to be dependent on the expression of secretory phospholipase A2 (sPLA2). In the present study, we investigated the possible involvement of Ca2+-independent phospholipase A2 (iPLA2) in the generation. The results showed that bromoenol lactone, a relatively selective iPLA2 inhibitor, significantly attenuated prostaglandin E2 generation induced by interleukin-1beta and dibutyryl cAMP in parallel with the inhibition of iPLA2 activity. However, the inhibitor did not affect sPLA2 release upon stimulation, activities of sPLA2 or cytosolic phospholipase A2, or Ca2+ ionophore-induced arachidonic acid liberation. These results suggest that prostaglandin E2 generation upon stimulation may be partially mediated by iPLA2 in addition to sPLA2.

Animals↗

Fungicidal activity of natural and synthetic sesquiterpene lactone analogs.

Fungicidal activity of 36 natural and synthetic sesquiterpene lactones with guaianolide, trans, trans-germacranolide, cis, cis-germacranolide, melampolide, and eudesmanolide carbon skeletons was evaluated against the phytopathogenic fungi Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Fusarium oxysporum, Botrytis cinerea, and Phomopsis sp. Dose-response data for the active compounds dehydrozaluzanin C, dehydrocostuslactone, 5alpha-hydroxydehydrocostuslacone, costunolide, and zaluzanin C are presented. A new 96-well microbioassay procedure for fast and easy evaluation of antifungal activity was used to compare these compounds with commercial fungicide standards. Some structure-activity conclusions are also presented.

Antifungal Agents↗

Sesquiterpene lactones of Centaurea scoparia.

From the ethanolic extract of the aerial parts of Centaurea scoparia four new sesquiterpene lactones, belonging to the guaiane class, were isolated and identified, together with a known germacranolide. The structures were elucidated by spectroscopic methods including one- and two-dimensional NMR studies. The relative configuration was deduced from the 2D NOESY spectra. CD spectroscopy of the chlorinated compounds was used for determination of their absolute configuration.

Journal Article↗